It is commonly assumed but not proven that microRNAs (miRNAs) and their targets coevolve. Under this assumption, miRNAs and targets from different species may interact adversely, resulting in reduced fitness. However, the strength of the adverse interactions may not be detectable because even outright deletions of miRNAs often manifest only subtle fitness effects. We tested and measured the strength of heterospecific interactions by carrying out transgenic experiments across Drosophila species by overexpressing the miR310s cluster of Drosophila melanogaster ( Dm310s ) and Drosophila pseudoobscura ( Dp310s ) in D. melanogaster . Flies overexpressing the heterospecific Dp310s are only one-third as viable as those overexpressing the conspecific Dm310s . The viability effect is easily detectable in comparison to the effect of the deletion of miR310s . The number of genes significantly misexpressed under the influence of Dp310s is 3–10 times greater than under Dm310s . Importantly, the numbers of predicted targets are similar between them. Expression analysis of the predicted target genes suggests that miRNAs may sometimes function to buffer fluctuations in the transcriptome output. After the buffering function has evolved, heterospecific combinations may cause adverse effects.